Despite the identification of the main components of the mammalian apoptotic pathway, the differences in the regulation of apoptosis in various primary cells remain surprisingly unexplored. Our unique ongoing contribution to the apoptosis field has been to define how different cell types set the apoptosis threshold to optimally matche their physiological functions and adapt to changing environments. Indeed, we believe that new paradigms of apoptosis regulation remain to be discovered in physiologically distinct cell types. Our lab has discovered that the apoptotic pathway is highly restricted in postmitotic cells such as neurons, cardiomyocytes, and myotubes, as compared to mitotic cells such as fibroblasts. While a strict regulation of apoptosis is critical for the long-term survival of postmitotic cells, mitotic cells need to maintain their ability to activate apoptosis rapidly as they can be at continual risk of becoming cancerous. Therefore, cells must efficiently balance the need for having a primed apoptotic pathway versus the risks associated with cell death. In fact, we have seen this best exemplified in embryonic stem (ES) cells which engage mechanisms that both prime the apoptotic pathway for rapid death in response to DNA damage, while also engaging cell survival mechanisms in response to mitochondrial damage. Thus, ES cells appear to have an exquisite capability to respond to the specific damage stimuli with mechanisms that ensure both genomic integrity and optimal survival. In this MIRA proposal, we wish to use both targeted and broad integrative approaches to examine the distinct mechanisms of apoptosis regulation and define their physiological importance in health and disease. Our focus is on the two extremes of the apoptosis control we identified: 1) Mechanisms that resistant apoptosis and promote survival after mitochondrial damage (e.g. our findings that the E3 ligase PARC mediates the degradation of cytosolic cytochrome c), and 2) Mechanisms that prime cells for rapid apoptosis (e.g. our discovery that Bax is maintained in an active state in stem cells). We will use primary neurons and human embryonic stem (hES) cells, conduct innovative screens and examine disease implications in models of cancer and neurodegeneration. In particular, we are excited that the MIRA opportunity would enable our ambitious plans to use the powerful capability of hES cells to define how the apoptotic machinery undergoes dynamic changes with cellular differentiation.